This project is focused on the identification of physiologically critical functions and mechanisms of action of NF-kB transcription factors and their regulators in health and disease. NF-kB is a family of related dimeric transcription factors that serve as primary intracellular mediators during innate and adaptive immune responses. In addition, and importantly, aberrant regulation of NF-kB plays a major role in inflammatory and autoimmune diseases as well as in numerous tumors. It is thus imperative to understand the functions and mechanisms of action of individual NF-kB factors and their regulators, as this will be required to devise appropriate strategies for therapeutic interventions aimed at curtailing aberrantly regulated NF-kB in a precisely targeted manner. To identify physiologic roles and mechanisms we make use of mouse models engineered to lack components of the NF-kB transcription factor family or their regulators, as well as models in which the NF-kB factors can be selectively activated. Our work is focused on so-called alternatively, but also classically activated NF-kB, but we are especially investigating the regulators Bcl-3 and IkBzeta. The alternative NF-kB activation pathway is normally initiated by a subset of TNF receptors. Bcl-3 and IkBzeta are atypical IkB family member that function as nuclear regulators of NF-kB activity. We previously discovered a critical role for Bcl-3 in the ability of dendritic cells to properly prime T cells to proliferate in response antigen, and thus to initiate a protective adaptive immune response to pathogens, such as to Toxoplasma gondii, a serious health risk in immune-compromised patients. In the absence of Bcl-3 in dendritic cells, mice succumb to this infection. Bcl-3 also has critical functions epithelial cells, such as in keratinocytes, where it helps to delimit hypersensitivity reactions. Of particular interest is our previous discovery that Bcl-3 is required in T cells to drive T-cell dependent autoimmune diseases, including experimental autoimmune encephalomyelitis, a model for Multiple Sclerosis, and T cell transfer-induced colitis, a model for Inflammatory Bowel Disease. In addition, we recently discovered that in contrast to its pro-tumorigenic role in B cells, it has a tumor-suppressive role in gut epithelial cells. Mice lacking Bcl-3 in these cells exhibited an increased tumor rate in a colitis inflammation-induced colon cancer model, highlighting the context-specific functions of the regulator. In FY2019 we have made significant progress towards our long-term goal to identify context-dependent functions of Bcl-3, its mechanisms of actions and its transcriptional targets. First, we collected wild-type and Bcl-3-deficient CD4 T cells from CD4-T cell transfer induced colitis mice and subjected them to bulk RNAseq analyses and are presently verifying potentially critical targets of Bcl-3. We also established an acute LCMV viral infection model to elucidate the role of Bcl-3. Using this model, we determined that Bcl-3 appears to have an important role in CD8 memory T cell development and we are in the process of analyzing single-cell RNAseq experiments. This opens the door towards understanding the overall functions, target genes and pathways controlled by Bcl-3 in a cell-type specific manner. Understanding what genes Bcl-3 controls in CD8 T cell memory formation is also vital in the context of tumor immunotherapy, where CD8 memory-like T cells, rather than effector T cells are thought to be specifically efficacious in long-term tumor suppression. In order to better understand the mechanisms of action of Bcl-3 we have made significant progress in identifying interacting proteins. In FY2019 we succeeded in generating and using mice in which Bcl-3 carries a small Tag that can be conditionally-attached in a cell-type specific manner. With these mice we are presently performing co-immunoprecipitation experiments to identify interacting proteins in T cells via MassSpec, specifically in the context of experimentally induced, T cell-dependent autoimmune diseases. In FY2019 we have also generated mice with conditional depletion of IkBz in T cells in the LCMV viral infection model and in keratinocytes in the Imiquimod-induced psoriatic inflammation model. Finally, we have generated mice in which the NF-kB factor RelB which is activated via the alternative pathway can be globally or conditionally ablated; we have used these mice to demonstrate a significant role for this NF-kB protein in EAE, a mouse model for Multiple Sclerosis. RelB turned out to have a negative effect on survival of mature oligodendrocytes, thus exacerbating pathogenesis.